The present invention relates to a novel method of purifying PrPres from a biological sample in order to use it for the qualitative and/or quantitative detection of PrPres in said sample.
Transmissible subacute spongiform encephalopathies are caused by non-conventional transmissible agents (NCTA), also called prions, the precise nature of which is still unknown at the present time. TSSE comprise essentially Creutzfeldt-Jakob disease (CID) in humans, scrapie in sheep and goats and bovine spongiform encephalopathy (BSE) in cattle; other encephalopathies have been revealed in mink or certain wild animals such as stag and elk.
The outcome of these diseases is inevitably fatal and no effective treatment is currently available.
In transmissible subacute spongiform encephalopathies, there is an accumulation of a host protein, PrP (or prion protein), in an abnormal form (PrPres), mainly in the central nervous system; PrPres copurifies with the infectiousness and its accumulation precedes the appearance of histological lesions. In vitro it is toxic to neuron cultures.
Two biochemical properties usually make it possible to distinguish between PrPres and normal PrP: PrPres is partially resistant to proteases and is insoluble in anionic surfactants.
To be able to detect the PrPres present in a sample, it is necessary to subject said sample to different operations in order to enrich it in PrPres, while eliminating the normal PrP, so that the PrPres can then be detected by any appropriate specific method without causing:
false positives due to the presence of normal PrP or other contaminants, or
false negatives due to an insufficient concentration of PrPres in the final biological sample.
A number of methods of isolating and/or purifying PrPres have been proposed for this purpose. They are essentially based on the method developed by Hilmert and Diringer (Nature, 1983, 306, 476-478) and generally involve an extraction with a detergent, differential ultracentriugations and a treatment with proteolytic enzymes (Multhaup G. et al., EMBO J., 1985, 4, 6, 1495-1501; Takahashi K et al., Microbiol. Immunol., 1986, 30, 2, 123-131; Hope J. et al., EMBO J., 1986, 5, 10, 2591-2597; Grathwohl K. U. D. et al., Arch. Virol., 1996, 141, 1863-1874; Kascsak R. J. et al., Immunol. Investig., 1997, 26, 259-268; R. E. Race et al., J. Gen. Virol., 1992, 73, 3319-3323; Doi et al., J. Gen. Virol., 1988, 69, 955-960; T. Muramoto et al., Am. J. Pathol., 1993, 143, 5, 1470-1479; Farquhar C. F. et al., Gen. Virol., 1994, 75, 495-504 and J. Gen. Virol., 1996, 77, 1941-1946). They have the disadvantage of comprising a large number of steps including several ultracentrifugations, which are cumbersome to carry out and result in cumulative losses of PrPres; these in turn lead to an insufficient sensitivity to obtain a high-quality detection threshold and quantification of the PrPres.
These various methods require research laboratory equipment and implementation times which are incompatible with use in the field, particularly in abattoirs.
Now, there is a need for rapid verification of the absence or presence of a transmissible subacute spongiform encephalopathy at the time when the animal is slaughtered.
Consequently the inventor set out to provide a method of purifying a biological sample in order to use it for a rapid and reliable detection of PrPres, said method being sufficiently simple to carry out that it can be used in the field, especially in abattoirs, and thereby meeting practical needs better than the methods of the prior art. In fact, the method according to the invention is:
simple to carry out,
reliable and
easy to interpret it increases the detection sensitivity threshold of PrPres by eliminating the false positives (normal PrP and other contaminants), and it eliminates the false negatives because it enables a substantial amount of PrPres to be obtained, in absolute terms, since it is possible to treat large amounts of biological material with a purification yield in excess of 80%; this is of particular value in abattoirs and produces samples in which PrPres is readily detectable with customary diagnostic tests.
The present invention relates to a method of purifying PrPres from a biological sample, characterized in that it comprises essentially:
(1) the incubation, for 30 seconds to 2 hours, preferably for 30 seconds to 10 minutes, at a temperature below 80xc2x0 C., of said biological sample with a buffer A comprising at least one surfactant in an amount of between a quarter and four times, preferably of between a quarter and one and a half times, the weight of the biological sample, and optionally prior, subsequent or simultaneous incubation with a protease, to form an opalescent to turbid micellar or lamellar suspension S1; under the temperature and quantity conditions mentioned above, whatever the surfactant or surfactant mixture may be, it does not solubilize most of the PrPres, which remains in suspension, whereas the normal PrP is solubilized, or even destroyed, if protease is added; said incubation is preferably carried out at a temperature below 50xc2x0 C., in the presence of an amount of surfactant of between a quarter and one and a half times the weight of the biological sample; according to the invention, the protease can in fact be added either before, after or simultaneously with the surfactant;
(2) the addition, to said micellar or lamellar suspension S1 obtained in (1), of a buffer B in an amount suitable for clarifying said suspension (for example by forming a microemulsion or a microsuspension), said buffer B consisting of a solvent or solvent mixture which does not solubilize the PrPres and has a dielectric constant of between 10 and 25; this gives a suspension S2 which is limpid to the naked eye;
(3) the centrifugation of the suspension S2 obtained in step (2); said centrifugation is carried out for example for 2 to 10 minutes at a speed below 20,000 g, preferably at a speed of between 3500 g and 17,500 g; the PrPres ends up in the centrifugation residue with a PrPres purification yield surprisingly of between 80 and 100%; advantageously the centrifugation time and speed can be adapted to give the same result, namely a PrPres purification yield of between 80 and 100%; and
(4) the solubilization of said residue in a buffer C comprising at least one surfactant, as defined in step (1), at a concentration of between 0.1% and 5%, preferably of between 0.25% and 1%, based on the volume of buffer C (w/v), and/or at least one chaotropic agent at a concentration of between 0.1 M and 8 M, at a temperature between room temperature and 100xc2x0 C., preferably equal to or greater than 80xc2x0 C.; under such temperature conditions, the above-mentioned surfactants, preferably ionic surfactants, and/or the chaotropic agents solubilize the PrPres.
Said steps (1) and (2) can be carried out simultaneously or successively; they are preferably carried out successively.
In one advantageous mode of carrying out said method, if the biological sample is a tissue or an organ, it is homogenized prior to step (1), for example by mechanical grinding in a homogenization buffer consisting of a neutral buffer such as water, or an isotonic buffer such as 5% glucose.
In another advantageous mode of carrying out said method, the temperature used in step (1) is between room temperature and 50xc2x0 C.; it is preferably 37xc2x0 C.
Buffer A preferably comprises a surfactant selected from the group consisting of:
anionic surfactants such as SDS (sodium dodecylsulfate), sarkosyl (lauroylsarcosine), sodium cholate, sodium deoxycholate or sodium taurocholate;
zwitterionic surfactants such as SB 3-10 (decyl sulfobetaine), SB 3-12 (dodecyl sulfobetaine), SB 3-14, SB 3-16 (hexadecyl sulfobetaine), CHAPS or deoxyCHAPS;
non-ionic surfactants such as C12E8 (dodecyl octaethylene glycol), Triton X100, Triton X114, Tween 20, Tween 80, MEGA 9 (nonanoylmethylglucamine), octylglucoside, LDAO (dodecyldirethylamine oxide) or NP40; or
surfactant mixtures such as a mixture of an ionic surfactant and a non-ionic surfactant, especially the mixture SDS/Tween 80 or the mixture sarkosyl/Triton X100, a mixture of two ionic surfactants, such as the mixture SDS/deoxycholate, or a mixture of an ionic surfactant and a witterionic surfactant.
In another advantageous mode of carrying out said method, buffer B is preferably selected from C3-C6 alcohols and alcohol mixtures with a mean theoretical dielectric constant of between 10 and 25. The following alcohols or alcohol mixtures are particularly preferred: butan-1-ol, butan-2-ol, 2-methylpropan-1-ol, isopropanol, isopropanol+pentanol, ethanol+hexanol, butanol+pentanol, etc.
In terms of the present invention, dielectric constant is understood as meaning the static dielectric constant xcex5, measured in static or relatively low frequency fields; it corresponds to the ratio of the electric displacement D to the electric field strength E when an electric field is applied to the solution at a temperature of between 293.15 and 298.15 K.
The dielectric constant of liquids, as defined above, is described more particularly in CRC Handbook of Chemistry and Physics (ed. David R. Lide, 75 th edition, 1994, CRC Press).
For a solvent mixture, mean theoretical dielectric constant is understood as the mean of the dielectric constants of each solvent, weighted by its proportion in the mixture.
Surprisingly the addition of buffer B in step (2) makes it possible to obtain purification yields in excess of 90% under low speed centrifugation conditions; it affords a significant reduction in the amount of final residue, while at the same time maintaining a high yield; advantageously the amount of final residue is preferably less than 10% of the initial weight of biological sample so as to be able to utilize it effectively in an immunoassay, whereas if only buffer A is added, the resulting conditions are those of the prior art, which necessitate an ultracentrifugation in order to obtain sufficient yields of PrPres for the purposes of detection.
It may be noted that purification yields in excess of 80% can also be obtained by varying the centrifugation time and speed: 2 to 10 minutes at a speed below 20,000 g or a period of time reduced in proportion to the increase in the number of g.
Preferably the ratio of the yield of PrPres in the solid phase to the amount of residue recovered after centrifigation of the suspension S2 is greater than 10 when the initial sample corresponds to 100 mg of brain.
As a further preference, buffer C used in step (4) comprises a chaotropic agent which is selected especially from the group consisting of urea and guanidine or a mixture thereof; it is also possible to use any other chaotropic agent.
The urea is preferably at a concentration of between 0.25 and 8 M and the guanidine is preferably at a concentration of between 0.1 and 6 M.
If buffer C is a mixture of at least one surfactant and at least one chaotropic agent, it is preferably selected from the group consisting of the following mixtures: a mixture of SDS and urea, a mixture of sarkosyl and urea, a mixture of deoxycholate and urea, a mixture of sarkosyl and guanidine or a mixture of sarkosyl, guanidine and urea Preferably, in the mixture of SDS and urea, the SDS is at a concentration of 0.25-1% and the urea is at a concentration of 0.25-6 M; in the mixture of sarkosyl and urea, the sarkosyl is at a concentration of between 0.25 and 1% and the urea is at a concentration of between 0.25 and 8 M; in the mixture of sarkosyl and guanidine, the sarkosyl is at a concentration of between 0.25 and 1% and the guanidine is at a concentration of between 0.5 M and 3 M, and in the mixture of sarkosyl, guanidine and urea, the sarkosyl is at a concentration of between 0.25 and 1%, the guanidine is at a concentration of between 0.5 M and 3 M and the urea is at a concentration of between 2 and 6 M.
Laemmli""s buffer (4% SDS, 0.1 M Tris-HCl pH 8, 5% sucrose and 2% xcex2-mercaptoethanol) can also be used, especially for western blotting.
The present invention further relates to a method of detecting PrPres in a biological sample, characterized in that it comprises:
treating said sample as defined above,
diluting the sample obtained, if necessary, and
detecting the PrPres by any appropriate analytical method, such as an immunological method (ELISA, western blotting), which produces a specific signal.
The above-mentioned dilution step makes it possible to neutralize buffer C to enable detection of the PrPres by an ELISA method; it is effected for example with a buffer comprising albumin to give a final albumin concentration of between 2 and 10% (w/v), or with a buffer based on 1% deoxycholate, for example.
A biological sample treated in this way contains an effective concentration of PrPres, so the latter can be detected directly in said sample by any analytical method, especially an immunological method.
As a variant, the present invention relates to a method of purifying PrPres from a biological sample, characterized in that it comprises essentially:
(1) the incubation, for 30 seconds to 2 hours, preferably for 30 seconds to 10 minutes, at a temperature below 80xc2x0 C., of said biological sample with a buffer A comprising at least one surfactant in an amount of between a quarter and four times, preferably of between a quarter and one and a half times, the weight of the biological sample, and optionally prior, subsequent or simultaneous incubation with a protease, to form an opalescent to turbid micellar or lamellar suspension S1; according to the invention, the protease is in fact added either before, after or simultaneously with the surfactant;
(2) the addition, to said micellar or lamellar suspension S1 obtained in (1), of a buffer B in an amount suitable for creating a phase separation, said buffer B consisting of a solvent or solvent mixture which does not solubilize the PrPres and has a dielectric constant of between 10 and 25;
(3) the centrifigation of the suspension obtained in step (2); said centrifugation is carried out for example for 2 to 10 minutes at a speed below 20,000 g, preferably at a speed of between 3500 g and 17,500 g; the PrPres ends up at the interface;
(4) the recovery of the film present at the interface;
(5) the resolubilization of said film with a buffer A without the addition of protease;
(6) the centrifugation of the suspension obtained in step (5) for 2 to 10 minutes at a speed below 20,000 g, preferably at a speed of between 3500 g and 17,500 g; the PrPres ends up in the centrifugation residue with a PrPres purification yield surprisingly of between 70 and 100%; advantageously the centrifugation time and speed can be adapted to give the same result, namely a PrPres purification yield of between 70 and 100%; and
(7) the solubilization of said residue in a buffer C comprising at least one surfactant, as defined in step (1), at a concentration of between 0.1% and 5%, preferably of between 0.25% and 1%, based on the volume of buffer C (w/v), and/or at least one chaotropic agent at a concentration of between 0.1 M and 8 M, at a temperature between room temperature and 100xc2x0 C., preferably equal to or greater than 80xc2x0 C.; under such temperature conditions, the above-mentioned surfactants, preferably ionic surfactants, and/or the chaotropic agents solubilize the PrPres.
The amounts of buffer B to be added to give a microemulsion, a micro-suspension or phase separation are established with the aid of a range of buffers B, as illustrated for butan-1-ol in FIG. 1; they can vary as a function of buffer A and the constituents selected for buffer B.
As a variant, the solubilization step (step (4) of the first method or step (7) of the second method) in said buffer C comprises heating at a temperature equal to or greater than 80xc2x0 C. for 5 to 10 minutes, followed by centrifugation, preferably for 2 to 10 minutes, at a speed below 20,000 g, preferably at a speed of between 3500 g and 17,500 g; in this case the PrPres is resolubilized and ends up in the supernatant; under such conditions, the sample obtained is particularly suitable for assaying the PrPres by an ELISA method.
The present invention further relates to a kit for treating a biological sample, characterized in that it comprises, in addition to a buffer for homogenizing said biological sample, appropriate amounts of buffer A, buffer B and buffer C, as defined above.
The present invention further relates to a PrPres detection kit, characterized in that it comprises appropriate amounts of a buffer for homogenizing the biological sample in which the PrPres is to be detected, appropriate amounts of buffer A, buffer B and buffer C, as defined above, and at least one appropriate anti-PrPres antibody.